Ls 


Nitrogen  Fixation  by 
Yeasts  db  Other  Fungi 
C.B.  L, 


CX) 


Main  Lib.    AGRIC,  OEPL 


NITROGEN    FIXATION   BY  YEASTS  AND  OTHER 

FUNGI 

AGRICU; 
LIBB/ 
ONIVE 

-—OF „ 

CALIF. 


BY 

CHARLES  B.  LIPMAN 


(FROM  THE  RESEARCH  LABORATORY  FOR  SOIL  CHEMISTRY  AND  BACTERI- 
OLOGY, UNIVERSITY  OF  CALIFORNIA) 


FROM 

JOURNAL  OF  BIOLOGICAL  CHEMISTRY 
VOL.  X,  No.  3,  OCTOBER,  1911 


•  'l:\iti   J  i'.. 


LIBRARY 
IVE 

Reprinted  from  THE  JOURNAL  OF  BIOLOGICAL  CHEMISTRY,  VOL.  X.IKfo.  3,  1<"^      QF 


NITROGEN  FIXATION  BY  YEASTS  AND  OTHER  FUNGI. 

BY  CHARLES  B.  LIPMAN. 

(From  the  Research  Laboratory  for  Soil  Chemistry  and  Bacteriology,  Univer- 
sity of  California.) 

(Received  for  publication,  July  28,  1911.) 

Since  the  epoch  making  discoveries  of  Hellriegel1  there  have 
followed  in  quick  succession  many  series  of  investigations  rela- 
tive to  the  fixation  of  atmospheric  nitrogen  by  living  organisms. 
Especially  brilliant  are  the  investigations  of  Winogradsky2  on 
Clostridium  pastorianum  and  those  of  Beyerinck3  on  the  Azotobac- 
ter  group,  and  it  is  unnecessary  here  to  go  into  a  review  of  the 
investigations  which  have  since  added  themselves  to  those,  for 
the  reader  is  doubtless  acquainted  with  the  painstaking  and  inter- 
esting researches  of  Gerlach  and  Vogel,  Kriiger  and  Schneidewind, 
J.  G.  Lipman,  Lohnis,  Christensen  and  a  host  of  others  to  whom  we 
are  indebted  for  information  on  the  subject  of  the  fixation  of 
atmospheric  nitrogen  by  soil  bacteria.  Suffice  it  to  say,  that 
the  researches  of  the  past  twenty-five  years  have  disclosed  facts 
which  point  emphatically  toward  the  conclusion  that  the  earth 
is  endowed  with  agencies  which,  at  least  in  part,  compensate  for 
the  losses  from  our  valuable  nitrogen  store  from  the  soil  which 
are  constantly  going  on.  From  the  earliest  of  these  researches 
it  appeared  that  the  power  of  living  organisms  to  assimilate  atmos- 
pheric nitrogen  was  limited  to  a  small  group  of  bacteria  of  which 
the  power  seemed  as  characteristic  and  distinct  as  did  the  power 
of  the  group  of  nitrifying  bacteria  to  change  ammonium  com- 
pounds to  nitrates,  but  the  later  investigations  soon  brought 
to  light  the  fact  that  many  bacteria  other  than  those  of  the  B. 
radicicola,  the  Azotobacter  and  the  Clostridium  groups  exhibited 
the  power,  more  or  less  pronounced,  of  fixing  atmospheric  nitro- 

]  Ueber  die  Stickstoffnahrung  der  Gramineen  und  Leguminosen,  1888. 
*Compt.  rend,  de  I'Acad.  des  Sri.,  1893. 
3Centralbl   f.  Bakt.,  ix,  2  Abt.,  p.  3,  1902. 

169 

272674 


170  Nitrogen  Fixation  by  Yeasts 

gen.  These  discoveries  led  further  to  a  study  of  other  organisms 
belonging  to  the  groups  of  yeasts,  molds  and  the  higher  fungi, 
in  a  search  for  those  among  them  which  possessed  the  peculiar 
physiological  power  to  fix  atmospheric  nitrogen.  These  studies 
have  resulted  in  both  positive  and  negative  results  frequently 
with  the  same  organisms  in  the  hands  of  different  investigators, 
and  the  present  status  of  the  question  is  still  unsettled  pending 
further  evidence  of  the  definite  and  constant  powers  of  the  fungi 
mentioned  to  fix  atmospheric  nitrogen.  It  was  therefore  with 
the  double  intention  of  making  a  further  contribution  to  our 
knowledge  of  the  nitrogen  fixing  powers  of  some  of  the  organisms 
already  studied  as  well  as  the  study  in  that  direction  of  organisms 
which  until  now,  so  far  as  the  writer  is  aware,  have  not  been 
experimented  with,  that  the  subjoined  investigations  were  under- 
taken. It  was  decided  to  make  a  study  of  the  nitrogen  fixing 
powers  of  some  of  the  true  yeasts,  "pseudo-yeasts/'1  Mycoderma 
varieties  and  some  of  the  common  molds,  among  them  Aspergillus 
miger,  Penicillium  glaucum,  Botrytis  cinerea  and  others.  Before, 
however,  going  into  the  detailed  description  of  the  experiments 
/carried  out  it  will  be  helpful  for  purposes  of  comparison  to  review 
briefly  the  several  researches,  which  have  been  carried  out  on  the 
nitrogen  fixing  powers  of  organisms  which  are  the  same  or  simi- 
lar to  those  employed  in  my  experiments. 

The  investigations  of  Jodin2  and  Hallier3  carried  out  as  early 
:as  the  sixties  of  the  last  century  led  them  to  believe  that  fungi 
were  possessed  of  the  power  to  fix  nitrogen.  Their  results  and 
opinions  on  this  matter  were  not  confirmed,  however  by  Woff 
and  Zimmerman.4 

Sestini  and  del  Torre  reported  some  investigations  in  1876 
which  seemed  to  have  a  doubtful  significance.  In  their  communi- 
cation they  make  mention,  however,  of  a  statement  by  Selmi  in 
which  the  latter  attributes  to  the  Mycoderma  forms  the  power 
to  produce  ammonia  from  atmospheric  nitrogen  in  the  presence 
of  nascent  hydrogen. 

1 A  term  employed  by  zymologists  to  designate  the  yeast-like  organisms 
which  do  not  form  spores. 

*Compt.  rend,  de  I'Acad.  des  Sci.,  Iv,  p.  612. 

*Zeitschr.  f.  Parasitenkunde,  i,  p.  129. 

4 Abstract  in  Jahresbeiicht  der  Agrikultur  Chemie,  xiii-xv,  p.  169 


Charles  B.   Lipman  171 

The  investigations  of  Loew  and  Nageli1  led  them  to  make  the 
statement  that  elementary  nitrogen  is  an  unsatisfactory  source 
of  that  element  for  molds.  Lawes,  Gilbert  and  Warrington2 
could  not  show  any  fixation  of  nitrogen  by  some  soil  molds  since 
analyses  of  soil,  in  which  these  molds  were,  showed  no  increase 
of  nitrogen. 

Winogradsky  has  reported  negative  results  in  experiments  with 
an  Aspergillus  species,  and  Czapek3  has  likewise  reported  nega- 
tive results  with  Aspergillus  niger  and  criticised  the  results  of 
Puriewitsch  and  Saida,  especially  the  latter,  on  the  ground  that 
the  amounts  of  nitrogen  fixed  may  have  been  due  to  errors  in  the 
nitrogen  determinations.  Gerlach  and  Vogel4  reported  negative 
results  with  a  yeast  experimented  with  by  them. 

To  all  of  these  doubtful  or  negative  results  on  the  fixation  of 
nitrogen  by  yeasts,  molds  and  other  fungi,  should  be  added  the 
results  of  investigations  very  recently  carried  out  by  Duggar  and 
Knudson5  at  Cornell  University  in  which  it  is  claimed  that  no 
nitrogen  fixation  was  observed,  except  to  a  very  slight  extent 
in  cultures  of  Aspergillus  niger. 

Opposed  to  these  negative  results  we  have  the  positive  results 
of  several  investigators  in  which  we  find  a  description  of  marked 
powers  of  fixing  nitrogen  attributed  to  the  yeasts  and  molds  experi- 
mented with.  Berthelot6  noted  gains  of  nitrogen  in  cultures  of 
Aspergillus  niger,  Alternaria  tennis  and  Gymnoascus,  but  only 
the  cultures  of  A  Uernaria  tennis  were  pure.  Puriewitsch, 7  however, 
worked  with  pure  cultures  of  Aspergillus  niger  and  Penicillium 
glaucum  and  in  addition  to  all  other  precautions  to  exclude  bac- 
teria from  these  cultures  he  added  some  phosphoric  acid  to  his 
culture  solution  which  latter  consisted  of  100  cc.  of  water,  0.4 
gram  of  monopotassium  phosphate,  0.4  gram  of  calcium  chloride, 
0.2  gram  of  magnesium  sulphate,  3  grams  of  tartaric  acid,  vary- 
ing amounts  of  dextrose  and  small  amounts  of  ammonium  nitrate. 

lSitzungsber.  d.  mathem.  physik.  Klasse  d.Akad.  Miinchen,  x,  p.  280. 
tJourn.  Chem.  Society,  Transactions,  xliii,  p.  208. 
*Beitr.  z.  chem.  Physiol.  und  PathoL,  ii,  p.  559,  1902. 
4Loc.  cit. 

'Abstract  in  Science,  Feb.  3,  1911,  of  a  paper  read  before  the  Botanical 
Society  of  America. 

6Chimie  vegetale  et  agricole,  i,  Paris,  1899. 
7Ber.  d.  deutsch.  bot.  Ges.,  xiii,  p.  339,  1895. 


172  Nitrogen  Fixation  by  Yeasts 

Gains  of  nitrogen  were  noted  in  all  cultures.  It  is  interesting 
however,  to  observe  in  his  results,  the  large  differences  in  the 
amounts  of  nitrogen  fixed  by  the  same  organism  in  duplicate  cul- 
tures. Saida1  not  only  confirmed  the  results  of  Puriewitsch  on 
Aspergillus  niger,  but  showed  that  a  distinct  nitrogen  fixing  power 
was  possessed  by  Mucor  stolonifer,  Endococcus  purpurascens, 
and  Phoma  betae.  No  gains  of  nitrogen,  however,  were  noted  by 
the  same  investigator  in  cultures  of  Acrostalagmus  cinnibarinus, 
Monilia  variabilis  and  Fusisporium  moschatum.  A.  Koch2  claims 
that  he  and  other  investigators  in  repeating  the  experiments  of 
Puriewitsch  and  Saida  could  not  obtain  any  fixation  of  nitrogen, 
but  calls  attention  to  the  fact  that  his  results  must  not  be  taken 
as  proof  of  the  questionable  purity  of  the  cultures  used  by  Purie- 
witsch and  Saida  nor  yet  of  any  error  in  the  work  of  the  last  named 
investigators  but  rather  to  a  change  in  the  character  of  the  organ- 
isms in  old  cultures  as  indeed  this  has  often  been  noted  in  cultures 
of  Azotobacter  which  after  long  standing  seemed  to  have  only  a 
feeble  nitrogen  fixing  power.  This  is  an  interesting  observation 
which  agrees  with*  a  similar  one  made  by  the  writer  in  the  experi- 
ments described  below. 

Frank3  showed  distinct  gains  of  nitrogen  in  work  carried  out 
with  different  species  of  Penicillium,  amounting  in  one  case  to 
3.5  mg.  of  nitrogen  in  65  cc.  of  nitrogen-free  sugar  solution  in 
an  incubation  period  of  ten  months.  Remy4  also  showed  fixation 
of  nitrogen  by  three  out  of  twenty-five  molds  which  he  tested 
and  among  these  was  Aspergillus  niger  which  fixed  10  mg.  of  nitro- 
gen on  20  grams  of  dextrose  as  a  source  of  energy.  To  these  must 
also  be  added  the  investigations  of  Ternetz5  who  found  in  working 
with  five  species  of  Phoma  that  the  latter  possessed  a  pronounced 
nitrogen  fixing  power,  noting  in  one  case  a  fixation  of  22  mg. 
of  nitrogen  per  gram  of  dextrose.  The  same  investigator  noted 
gains  of  nitrogen  also  in  cultures  of  Penicillium  glaucum  and  Asper- 
gillus niger  amounting  in  the  latter  case  to  2.71  mg.  nitrogen  per 
gram  of  dextrose. 

lBer.  d.  deutsch.  bot.  Ges.,  xix,  p.  107,  1901. 
2Handbuch  der  technischen  Mykologie,  Jena,  iii,  1907. 
*Landw.  Jahrb.,  xxi,  p.  7,  1892;  Bot.  Zeitung,  li,  p.  146,  1893. 
4  Verh.  d.  Ges.  deutsch.  Naturf.  und  Aerzte,  Ixxiv,  i,  p.  221,  1902. 
*Ber.  d.  deutsch.  bot.  Ges.,  xxii,  p.  267;  Jahrb.  f.  wissen.  Botanik,  xliv, 
1907,  p.  353-408. 


Charles  B.  Lipman  173 

Gains  of  nitrogen  were  also  noted  by  Heinze1  in  cultures  of  yeasts 
in  their  spore  forming  stages  and  in  cultures  of  lichens  in  the 
gonidium  form.  There  should  also  be  mentioned  here  the  experi- 
ments of  Gerlach  and  Vogel2  in  which  they  found  a  mold  to  pos- 
sess the  power  of  increasing  the  nitrogen  content  of  the  culture 
from  2.8  mg.  to  5.1  mg.  This  increase  is  attributed  by  Gerlach 
and  Vogel  to  experimental  error,  but,  as  Lohnis  well  remarks  in 
reviewing  this  matter,  the  other  data  given  in  the  same  investi- 
gations do  not  support  such  a  view. 

In  a  series  of  very  carefully  conducted  experiments  in  which 
every  possible  precaution  was  taken  to  prevent  absorption  of 
ammonia  and  amido  compounds  from  the  air  by  the  culture  solu- 
tions, Frohlich3  showed  very  considerable  and  definite  gains  of 
nitrogen  in  cultures  of  fungi  obtained  from  their  growths  on  dead 
parts  of  plants.  These  fungi  belonged  to  the  hyphomycetes, 
and  one  of  them  Macrosporium  commune  fixed  on  the  average 
as  much  as  8.92  mg.  of  nitrogen  per  gram  of  dextrose  used.  In 
addition  to  these  results  the  same  investigator  confirmed  the  results 
of  others  above  noted  with  respect  to  the  nitrogen  fixing  powers 
of  Penicillium  glaucum  and  Aspergillus  niger.  Latham4  further 
confirms  the  results  of  Berthelot,  Puriewitsch,  Saida,  Remy, 
Ternetz,  Frohlich  and  others  above  mentioned,  in  his  experiments 
with  Aspergillus  niger. 

Of  particular  interest  in  connection  with  the  writer's  results 
are  those  of  Zikes5  who  describes  a  torula  form,  isolated  from  laurel 
leaves  and  named  by  him  Torula  wiesneri  which  possesses  a 
power  of  nitrogen  fixation  equal  to  2.3  to  2.4  mg.  per  gram  of  glu- 
cose used.  The  experiments  of  Lohnis  and  Pillai6  show  only 
a  slight  nitrogen  fixing  power  for  a  torula  form  employed  by  them, 
but  a  larger  fixation  of  nitrogen  by  Dematium  pullulans. 

lCentralbl.  f.  Bakt.,  x,  2  Abt.,  p.  675;  xii,  p.  357. 
*Beitr.  z.  chem.  Physiol.  u.  Path.,  ii,  1907. 
*Jahrb.  f.  wissen.  Botanik,  xlv,  p.  256,  1907. 
4 Bull.  Torrey  Bot.  Club,  xxxvi,  p.  235,  1909. 
*Sitzungsber.  Akad.  Wien,  math-naturw .  KL,  cxviii,  p.  1091. 
8 Lohnis  and  Pillai:  Centralbl.  f.  Bakt.,  xx,  2  Abt.,  p.  799. 


174  Nitrogen  Fixation  by  Yeasts 

EXPERIMENTAL. 

The  material  tested  in  these  experiments  consisted  of  seven 
species  of  saccharomyces,  six  varieties  of  "  pseudo-yeasts,"1 
one  mycoderma  (Mycoderma  vim)  and  three  molds.  The  cultures 
employed  were  kindly  given  to  the  writer  by  Prof.  F.  T.  Bioletti 
of  the  California  Agricultural  Experiment  Station  to  whom  I 
desire,  here,  to  express  my  sincere  thanks.  The  cultures  were 
all  examined  microscopically  and  appeared  to  be  pure  cultures 
of  the  organisms  named  in  the  tables. 

Series  I. 

A  culture  solution2  was  prepared  and  distributed  in  100  cc. 
portions  in  500  cc.  Erlenmeyer  flasks  and  sterilized  in  the  auto- 
clave. Each  liter  of  solution  consisted  of  the  following: 

15.0    grams  mannite. 
0.2    gram  dipotassium  phosphate. 
0.2    gram  magnesium  sulphate. 
0.02  gram  calcium  chloride. 

3  drops  of  a  10  per  cent  solution  of  ferric  chloride. 
1000  cc.  tap  water. 

The  solution  was  rendered  slightly  alkaline  to  phenolphthalein 
by  means  of  sodium  hydrate.  The  solutions  were  carefully  inoc- 
ulated to  prevent  contamination,  by  means  of  a  sterile  platinum 
loop  and  placed  in  the  incubator  at  26°-28°  C.  for  one  month, 
after  which  they  were  transferred  to  Kjeldahl  digestion  flasks, 
30  cc.  concentrated  sulphuric  acid  added  and  boiled  on  the  diges- 
tion shelf  until  frothing  ceased3;  then  about  12  grams  of  a  mixture 
of  K2S04,  FeS04  and  CuS04  (in  the  proportion  of  10  to  1  to  i)  were 
added  and  the  digestion  continued  for  another  hour  or  more.  When 
cool  the  digested  solutions  were  transferred  to  copper  distilling 

1  These  organisms  are  described  by  Holm:  Bull.  No.  197,  Cal.  Expt.  Sta- 
tion. 

2 Used  byLipman  and  Brown:  New  Jersey  Agr.  Expt.  Station,  Bulletin 
No.  210. 

3  The  method  for  the  nitrogen  determination  used  is  given  in  detail  because 
it  presents  some  new  modifications  which  allow  of  rapid  and  accurate  work, 
as  shown  by  tests  in  experiments  which  will  be  published  shortly. 


Charles  B.   Lipman 


175 


flasks,  diluted  to  proper  volume,  an  excess  of  lye  added,  also 
some  powdered  zinc  to  prevent  bumping  and  the  ammonia  dis-' 
tilling  over  was  caught  in  ^  HC1.  The  excess  of  the  latter 
was  titrated  against  ^  NH4OH;  and  the  amount  of  nitrogen 
fixed  calculated  in  milligrams.  Several  sterile  controls  were  run 
with  each  series.  The  results  of  the  first  series  follow: 

TABLE  I. 


NO. 

NAME 

NITROGEN 
FOUND 

NITROGEN 
FIXED 

1 

2 
3 

Saccharomyces  apiculatus  
Saccharomyces  ellipsoideus,  champagne  
Saccharomyces  cerevisioe,  carlsbergensis 

mg. 

1.75 

1.87 
1  87 

mg. 

0.84 
0.96 
0  96 

4 
5 
6 

7 

Saccharomyces  ellipsoideus,  Steinberg  
Saccharomyces  cerevisioe,  Distillery  Ra  
Saccharomyces  ellipsoideus,  Bioletti  
Mycoderma  vini  

1.09 
1.04 
1.01 
1  40 

0.18 
0.13 
0.10 
0  49 

8 
9 
10 

Pseudo  yeast,  Tulare  No.  46a  
Pseudo  yeast,  Tulare  No.  46b  
Pseudo  yeast,  Tulare  No.  45b  

1.26 
2.38 
0  91 

0.35 
1.47 
0  00 

11 
12 

Pseudo  yeast,  Tulare  No.  28a  
Pseudo  yeast,  Tulare  No.  26 

1.12 
1  26 

0.21 
0  35 

13 

Pseudo  yeast,  Tulare  No.  37  

0  95 

0  04 

14 

Aspergillus  niger  

1  40 

0  49 

Every  culture  seems  to  show  an  increase  of  nitrogen  except 
nos.  10  and  13,  but  it  may  also  be  probable  that  the  amounts 
fixed  in  nos.  4,  5,  6  and  11  may  lie  within  the  limits  of  error,  and 
possibly  even  nos.  8  and  12  may  be  included  in  this  list.  Never- 
theless, there  is  a  distinct  fixation  of  nitrogen  in  nos.  1,  2,  3,  7,  9 
and  14,  or  in  six  out  of  fourteen  cultures,  and  it  is  interesting 
to  note  that  of  these  six,  three  are  true  yeasts,  one  a  mycoderma, 
one  a  pseudo  yeast  and  one  a  mold.  The  largest  amount  fixed 
in  this  series  was  that  in  no.  9  by  the  "  pseudo  yeast"  called 
Tulare  no.  46b. 

It  would  seem  therefore  that  these  results  are  a  further  confirma- 
tion of  those  of  Saida,  Puriewitsch,  Ternetz,  Frohlich,  Zikes  and 
others  above  mentioned,  and  it  is  especially  interesting  to  note 
that  the  organism  in  the  series  above  given  which  showed  the 
largest  fixation  of  nitrogen  is  very  much  like  the  Torula  wiesneri 


176  Nitrogen  Fixation  by  Yeasts 

with  which  Zikes  worked  and  which  fixed   considerably  more 
nitrogen. 

That  fixation  should  further  have  taken  place  in  cultures  repre- 
senting the  various  classes  of  organisms  experimented  with, 
confirms  the  writer  in  the  belief  which  he  has  held  ever  since  these 
investigations  were  begun  that  the  power  of  fixing  atmospheric 
nitrogen  though  perhaps  not  a  universal  one  among  the  lower 
plants  is  yet  a  very  widespread  power  among  the  fungi.  The 
fixation  of  nitrogen  by  true  yeasts  is,  so  far  as  the  writer  is  aware, 
the  first  one  described  as  such  in  the  literature  on  the  subject  and 
adds  another  class  of  organisms  to  the  now  rapidly  growing  list 
of  those  which  seem  to  be  possessed  of  that  power.  It  must 
also  be  mentioned  here  that  the  culture  solution  used  above 
while  a  very  good  one  for  organisms  of  the  Azotobacter  group  is 
not  necessarily  a  satisfactory  one  for  the  organisms  studied  and 
was  only  employed  because  of  the  fact  that  so  many  different 
organisms  were  used  and  further  obviously  that  no  information 
is  available  as  to  what  constitutes  a  good  medium  for  nitrogen 
fixation  for  each  class  of  organisms  tested.  It  is  possible,  for 
example,  that  the  alkaline  reaction  of  the  culture  solution  which 
is  so  necessary  to  the  fixation  of  nitrogen  by  Azotobacter  may  be 
rather  a  hindrance  than  otherwise  to  the  nitrogen  fixing  powers 
of  Aspergillus  niger  or  even  the  yeasts.  Despite  that,  however, 
we  have  evidence  of  a  power  of  nitrogen  fixation,  more  or  less 
pronounced,  in  each  of  these  classes  of  organisms. 

Series  II. 

Owing  to  the  fact  that  the  zymologist  values  dextrose  so  highly 
as  a  medium  for  fermentation  by  yeasts,  it  was  decided  to  arrange 
a  series  like  the  preceding  but  to  substitute  dextrose  for  mannite. 
The  solutions  were  distributed  in  100  cc.  portions  in  500  cc. 
Erlenmeyer  flasks  as  described  in  the  first  series,  and  after  the 
requisite  sterilization  and  cooling  were  inoculated  from  the  beer 
wort  cultures  of  the  organisms  tested.  The  incubation  was 
carried  out  in  the  same  manner  as  in  the  preceding  series,  after 
which  the  solutions  were  analyzed  for  nitrogen  according  to  the 
modification  of  the  Kjeldahl  method  above  described.  The 
results  of  the  analyses  follow: 


Charles  B.   Lipman 


177 


TABLE  II. 


NO. 

NAME 

NITROGEN 
FOUND 

NITROGEN 
FIXED 

1 

Saccharomyces  apiculatus 

mg. 

3  37 

mg. 

0  92 

2 
3 

Saccharomyces  ellipsoideus,  champagne  
Saccharomyces  cerevisioe,  carlsbergensis  

2.69 
4.19 

0.24 
1  74 

4 

Saccharomyces  ellipsoideus,  Steinberg    .  .    . 

3  43 

0  98 

5 

Saccharomyces  cerevisioe,  Distillery  R2 

3  99 

1  54 

6 

Saccharomyces  ellipsoideus,  Bioletti  

2.63 

0.18 

7 
8 

Saccharomyces  ellipsoideus,  Burgundy  
Mycoderma  vini                .    ... 

2.59 
4  24 

0.14 
1  79 

9 

Pseudo  yeast,  Tulare  No.  46a 

4  73 

2  28 

10 

Pseudo  yeast   Tulare  No  46b 

3  71 

1  26 

11 

Pseudo  yeast,  Tulare  No.  45b  

2  52 

0  07 

12 

Pseudo  yeast,  Tulare  No.  28a   . 

4  54 

2  09 

13 

Pseudo  yeast,  Tulare  No.  26  

3.50 

1.05 

14 

Aspergillus  niger  

4  68 

2  23 

15 

Penicillium  glaucum              

3  15 

1  70 

A  glance  at  Table  II  shows  the  influence  of  the  kind  of  medium 
on  the  amount  of  nitrogen  fixed.  Not  only  does  dextrose  allow 
of  a  much  larger  fixation  of  nitrogen  by  the  same  organisms  which 
showed  fixation  in  mannite  solutions  but  it  allows  other  organisms 
to  fix  nitrogen  which  showed  but  a  small  nitrogen  fixing  power 
or  none  at  all  in  the  other  medium.  This  is  a  factor  which  can- 
not be  overlooked  in  considering  the  practical  phases  of  nitrogen 
fixation  as  related  to  the  nitrogen  supply  for  plants  in  the  uni- 
verse. Here  again,  it  would  appear,  the  pseudo  yeasts  are  more 
efficient  at  nitrogen  fixation,  when  showing  that  power  at  all, 
than  the  true  yeasts.  For  Aspergillus  niger  we  find  dextrose  to 
be  far  superior  to  mannite  as  a  source  of  energy,  and  we  find  it 
to  have  a  power  of  nitrogen  fixation  in  the  medium  which  corre- 
sponds closely  to  that  exhibited  by  the  same  organism  in  the  hands 
of  other  investigators  above  mentioned.  Penicillium  glaucum, 
too,  manifests  a  definite  power  of  nitrogen  fixation  in  dextrose 
solutions  and  this  again  corresponds  to  the  results  obtained  by 
other  investigators  whose  work  is  above  reviewed. 

We  find  thus  that  at  least  eleven  of  the  fifteen  organisms  above 
tested  show  more  or  less  pronounced  powers  of  fixing  nitrogen. 
Owing  to  the  higher  content  of  nitrogen  in  the  tap  water  used  at 
this  time  and  also  the  higher  content  of  nitrogen  in  the  lye  and 


178  Nitrogen  Fixation  by  Yeasts 

acid  used  in  the  nitrogen  determinations,  we  find  a  much  higher 
blank  here,  but  several  blanks  were  analyzed  and  the  close 
agreement  between  them  showed  the  results  above  given  to  be 
trustworthy.  It  should  be  mentioned  here  that  the  sterile  blanks 
in  all  these  series  were  not  merely  analyzed  from  the  original  solu- 
tion but  were  inoculated  like  the  cultures,  then  sterilized  and  incu- 
bated side  by  side  with  the  cultures  for  the  same  length  of  time.  It 
is  interesting  to  note  here  that  the  amount  of  visible  growth  cannot 
be  directly  correlated  with  the  amount  of  nitrogen  fixed,  for  sev- 
eral of  the  cultures  which  appeared  to  have  made  only  a  small 
amount  of  growth  showed  quite  a  considerable  fixation  of  nitrogen. 
I  presume  that  this  has  been  observed  by  other  investigators  work- 
ing on  this  problem  and  is  probably  due  to  the  fact  that  some 
of  the  nitrogen  compounds  produced  are  soluble  and  diffusible  and 
therefore  give  no  visible  evidence  of  their  presence.  It  is  hardly 
necessary  to  add  here  that  despite  the  favorable  constitution  of 
the  medium  employed  it  is  not  nearly  so  favorable  for  the  growth 
of  the  organisms  tested  as  the  beer  wort  in  which  the  stock  cul- 
tures were  kept  as  can  be  noticed  particularly  in  the  cultures  of 
Aspergillus  niger  and  Penicillium  glaucum  where  the  membranes 
formed  in  the  dextrose  solution  are  very  thin  and  light  in  color  and 
the  spore  production  much  smaller,  than  in  the  beer  wort  cultures. 

Series  III. 

As  explained  above  the  culture  solutions  employed  in  the  pre- 
ceding series  were  prepared  with  tap  water  and  the  necessary  sugar 
and  salts  added.  It  appeared  to  the  writer  that  a  more  rigid 
test  of  the  power  to  fix  nitrogen  possessed  by  the  organisms  in 
question  should  be  made.  It  seemed  desirable  to  see  if,  like  the 
nitrogen  fixing  bacteria  of  the  Azotobacter  group,  they  had  the 
power  to  fix  nitrogen  in  nitrogen-free  solutions  or  solutions  which 
are  practically  nitrogen-free.  It  was  also  thought  desirable  to 
test  the  comparative  values  of  mannite  and  four  of  the  sugars  in 
such  nitrogen-free  solutions  as  sources  of  energy  for  the  organisms. 
To  that  end  solutions  like  the  one  described  above  were  prepared 
but  distilled  water,  free  from  ammonia,  was  substituted  for  tap 
water.  The  salts  employed  being  chemically  pure  and  used  only 
in  small  quantities  as  noted,  could  not  contain  more  than  a  trace 


Charles  B.   Lipman  179 

of  nitrogen.  The  sugars  employed  besides  mannite  were  dex- 
trose, maltose,  lactose  and  saccharose.  These  were  all  chemi- 
cally pure  and  thus  the  solutions  when  made  up  could  only  con- 
tain traces  of  combined  nitrogen. 

A  change  in  the  method  of  preparing  the  cultures  in  addition 
to  the  above  should  also  be  noted  here.  Twenty,  instead  of  15, 
grams  of  sugar  were  added  to  each  liter  of  solution.  The  latter 
was  distributed  in  50  cc.  portions  in  250  cc.  Erlenmeyer  flasks 
each  of  which  therefore  contained  1  gram  of  mannite  or  sugar. 
The  inoculations  and  incubation  were  carried  out  as  in  the  pre- 
ceding series  except  that  the  cultures  in  the  maltose,  lactose  and 
saccharose  solutions  were  incubated  for  twenty-five  days  instead 
of  one  month  as  were  all  the  others.  The  superior  nature  of  the 
tap  water  as  compared  with  the  distilled  water  was  seen  early  in 
the  incubation  period.  The  growth  in  the  distilled  water  cultures 
was  much  slighter  and  this  was  particularly  noticeable  in  the  case 
of  the  molds.  The  results  of  the  nitrogen  determinations  fol- 
low, all  arranged  in  one  table  so  that  the  various  sugars  may  be 
readily  compared. 

The  results  in  Table  III  show  clearly  that  every  one  of  the 
organisms  tested  possesses  a  power,  more  or  less  marked,  of  fix- 
ing atmospheric  nitrogen.  In  some  cases  that  power  seems  to 
be  so  slight  indeed  as  to  be  negligible  but  in  most  cases  it  is  very 
distinct  and  definite.  The  next  striking  fact  which  presents  itself 
for  consideration  in  an  examination  of  the  foregoing  table  is  the 
great  difference  in  the  nitrogen  fixing  power,  of  the  several  organ- 
isms tested,  in  the  different  media.  While  mannite  seems  to  have 
been  the  most  favorable  source  of  energy  for  the  largest  number 
of  organisms  tested,  some  of  the  sugars  employed  allowed  of  the 
fixation  of  nitrogen  by  organisms  which  did  not  fix  any  nitrogen 
at  all  in  mannite  solutions. 

The  highest  amounts  of  nitrogen  fixed  were  quite  considerable 
and  compare  well  with  the  amounts  fixed  by  pure  cultures  of  Clos- 
tridium  pastorianum  and  some  of  the  less  vigorous  species  of  the 
Azotobacter  group.  We  find  here  again  in  the  dextrose  and  lac- 
tose solutions  a  confirmation  of  the  work  of  other  investigators 
above  mentioned  with  respect  to  Aspergillus  niger,  and  in  the 
mannite  solutions  with  respect  to  Penicillium  glaucum.  While 
the  amounts  fixed  are  in  most  cases  not  as  large  in  these  distilled 


i8o 


Nitrogen  Fixation  by  Yeasts 

TABLE  III. 


NO. 
1 

2 
3 
4 
5 
6 
7 

8 
9 

10 
11 
12 

13 
14 
15 

NAME 

DEXTROSE 

MANNITE 

MALTOSE 

LACTOSE 

SACCHA- 
KOSE 

Nitrogen 

Nitrogen 

Nitrogen 

Nitrogen 

Nitrogen 

1 

I 

| 

1 

1 

I 

Q 

mg. 

1.79 
0.66 
0.73 
1.16 

1.34 

3.54 
1.51 

1.05 
0.97 
0.84 

1.08 
2.13 
0.85 
0.63 

TJ 

§ 

<« 

T3 

a 

mg. 

0.35 
0.35 
0.14 
0.35 
0.49 
0.21 
0.42 

0.07 
0.21 
0.21 

0.21 

Saccharomyces   apiculatus.. 
Saccharomyces    ellipsoideus, 
champagne             

mg. 
1.05 

1.78 
1.88 
1.26 
1.26 
1.21 

1.12 
1.71 

1.21 
1.24 
1.40 

1.82 
1.75 
1.12 
1.26 

mg. 

0.66 
0.76 
0.14 
0.14 
0.09 

0.59 
0.09 
0.12 
0.28 

0.70 
0.63 

0.14 

mg. 
2.38 

0.84 
0.98 
1.40 
1.12 
0.78 

2.10 
0.84 

1.16 

3.78 
0.70 

1.54 
0.98 
2.80 
3.22 

mg. 

1.54 

0.14 
0.56 
0.28 

1.26 

0.32 
2.94 

0.70 
0.14 
1.96 
2.38 

mg. 

0.70 
0.50 
0.90 
0.70 
0.70 
1.05 

0.35 
1.36 

0.70 
0.94 
1.58 

0.41 
0.59 
0.75 
0.70 

mg. 

0.20 

0.35 
0.66 

0.24 

0.88 

0.05 

mg. 

0.99 

0.36 

0.54 

2.74 
0.71 

0.25 
0.17 
0.04 

0.28 
1.33 
0.05 

mg. 

0.98 
0.98 
0.77 
0.98 
1.12 
0.84 

1.05 
0.63 

0.70 

0.84 
0.84 

0.84 
0.63 

Saccharomyces       cerevisioe, 
carlsergensis 

Saccharomyces   ellipsoideus, 
Steinberg                 

Saccharomyces       cerevisioe, 
Distillery   R2  
Saccharomyces    ellipsoideus, 
Bioletti 

Saccharomyces   ellipsoideus, 
Burgundy           

Mycoderma  vini 

Pseudo    yeast,    Tulare    No. 
46a                     

Pseudo   yeast,    Tulare    No. 
46b             

Pseudo    yeast,    Tulare    No. 
28a                           

Pseudo    yeast,    Tulare    No. 
26                    

Aspergillus  niger 

Penicillium  glaucum 

Botrytis  cinerea  

water  media  as  in  the  tap  water  media,  they  are  in  many  cases 
definite  enough  to  remove  any  doubt  that  these  organisms  are 
possessed  of  the  power  of  fixing  atmospheric  nitrogen,  even  if 
amounts  below  0.3  mg.  nitrogen  are  to  be  attributed  to  experi- 
mental error  which  indeed  the  writer  very  seriously  doubts. 
There  were  slight  losses  of  nitrogen  from  some  of  the  culture  solu- 
tions which  can  hardly  be  explained  in  any  other  way  than  by 
attributing  them  to  experimental  error.  It  is  very  striking  that 
organisms  which  in  their  natural  habitats  are  accustomed  to  draw- 
ing their  nitrogen  supply  from  a  plentiful  store  of  that  element 


Charles  B.   Lipman  181 

can  be  made  to  fix  atmospheric  nitrogen  in  nitrogen-poor  or  nitro- 
gen-free solutions  with  one  of  the  sugars  or  mannite  as  a  source 
of  energy.  Maltose  and  saccharose  do  not  seem  to  be  nearly  as 
well  suited  to  the  growth  and  development  of  the  organisms 
tested  as  lactose,  dextrose  and  mannite,  the  last  seeming  to  be  the 
most  favorable  when  the  organisms  are  considered  as  a  whole. 
The  largest  fixation  of  nitrogen  was  2.94  mg.  per  gram  of  mannite 
fixed  by  Pseudo-yeast  Tulare  no.  46b.  The  next  largest  2.74 
mg.  by  the  Burgundy  wine  yeast  per  gram  of  lactose  and  the  next 
largest  2.38  mg.  per  gram  mannite  by  Botrytis  cinerea.  Several  other 
considerable  amounts  were  fixed  by  other  organisms  in  mannite 
and  lactose  solutions  which  two  seem  to  be  the  best  suited  for  the 
fixation  of  large  amounts  of  nitrogen  in  distilled  water  cultures. 

GENERAL  DISCUSSION. 

A  careful  consideration  of  the  data  above  given  brings  further 
confirmation  of  the  work  of  other  investigators  to  the  effect  that 
the  power  of  fixing  atmospheric  nitrogen  is  possessed  by  many 
of  the  lower  organisms  which  differ  widely  in  their  character. 
Though  the  amounts  fixed  by  them,  as  shown  above,  are  not  as 
large  as  those  fixed  by  B.  radicicola  in  conjunction  with  the  legu- 
minous plants  nor  yet  as  large  as  those  fixed  by  the  more  vigorous 
species  of  the  Azotobacter  group,  they  are  none  the  less  definite 
and  considerable.  To  the  list  of  organisms  which  can  fix  atmos- 
pheric nitrogen  as  shown  by  former  investigations  may  now  be 
added  the  true  yeasts  and  the  "pseudo  yeasts,"  besides  Botrytis 
cinerea,  an  organism  whose  parasitic  nature  would  seem  to  have 
deprived  it  of  any  nitrogen  fixing  power  whatever.  This  in  itself 
is  a  very  interesting  and  striking  fact.  The  fixation  of  nitrogen 
seems  to  have  been  made  easier  for  the  organisms  in  tap  water 
solutions  than  in  distilled  water  solutions  owing  to  the  small  amounts 
of  combined  nitrogen  present  in  the  former.  The  nitrogen  fixed 
would,  in  many  cases,  seem  to  have  been  of  a  soluble  nature  since 
considerable  fixation  was  often  noted  in  solutions  where  the  growth 
would  not  seem  to  indicate  it.  The  conclusions  of  Duggar  and 
Knudson1  are  therefore  not  supported  by  the  investigations  above 
described.  An  effort  is  now  being  made  by  the  writer  to  carry 

1  Science,  N.  S.,  xxxiii,  p.  191,  1911. 


1 82  Nitrogen  Fixation  by  Yeasts 

out  some  experiments  on  the  fixation  of  nitrogen  by  fungi  similar 
to  those  with  which  Duggar  and  Knudson  worked  and  it  is  hoped 
that  the  results  may  be  available  for  publication  in  the  near  future. 
The  objections  of  Czapek  to  the  work  of  Puriewitsch  and  Saida 
would  not  seem  to  be  valid  in  view  of  the  writer's  experiments, 
since  the  method  of  nitrogen  determination  employed  as  above 
described  has  been  carefully  tested  in  my  laboratory,  allows  of 
a  close  agreement  between  duplicate  series  of  determinations,  and 
has  given  most  satisfactory  results  in  other  phases  of  microbio- 
logical work. 

SUMMARY   OF   RESULTS. 

1.  Of  eighteen  organisms,  including  yeasts,  pseudo  yeasts  and 
molds,  tested  nearly  all  show  a  more  or  less  pronounced  power  of 
fixing  atmospheric  nitrogen. 

2.  Tap  water  sugar  solutions  are  better  suited  for  nitrogen 
fixation  by  the  organisms  tested  than  distilled  water  solutions. 

3.  Mannite  and  lactose  solutions  are  far  superior  to  dextrose, 
saccharose  and  maltose  solutions  for  these  organisms  in  distilled 
water,  but  dextrose  is  the  best  in  tap  water  solutions.     Maltose 
is  the  most  unsatisfactory. 

4.  The  highest  amount  of  nitrogen  fixed  was  2.94  mg.  per  gram 
of  mannite  by  pseudo  yeast  Tulare  no.  46b  in  distilled  water  man- 
nite  solution. 

5.  The  results  of  other  investigators  with  reference  to  the  nitro- 
gen fixing  powers  of  Aspergillus  niger  and  Penicillium  glaucum 
are  confirmed. 

6.  Botrytis  cinerea,  a  parasitic  fungus,  has  been  found  for  the 
first  time,  so  far  as  the  writer  is  aware,  to  possess  a  nitrogen  fix- 
ing power. 

My  thanks  are  due  my  former  assistant,  Mr.  J.  A.  McKeen, 
for  valuable  assistance  in  making  the  nitrogen  determinations. 


2KS£^%«"», 


JUL 


10m-l«,'23 


J 


ivi  alters 

Syracuse,  N.  Y. 
PAT.  JAN.  21,  1908 


272674 


UNIVERSITY  OF  CALIFORNIA  IvIBRARY 


